Reaching the Monophyly: Re-Evaluation of the Enigmatic Species Tenuibiotus hyperonyx (Maucci, 1983) and the Genus Tenuibiotus (Eutardigrada)

Simple Summary The tardigrade species Tenuibiotus hyperonyx (Maucci, 1982) was described forty years ago from the highest mountain of the Sella Group within the Dolomites (Italy), and so far, it is known only from its type locality. Due to the morphological characters of the claws, it has been included in the genus Tenuibiotus (Pilato & Lisi, 2011). By conducting the integrative and phylogenetic analyses, we pinpointed the phylogenetic position of the species, which turned out to be positioned within the genus Diaforobiotus Guidetti et al., 2016. As the phenotypic characters of the examined species fit the diagnosis of this genus, we proposed a new combination, Diaforobiotus hyperonyx (Maucci, 1982) comb. nov. Furthermore, we looked more closely for the morphological diversity noted within the genus Tenuibiotus, clarifying phenotypic uncertainties regarding Tenuibiotus willardi (Pilato, 1977) and Tenuibiotus bozhkae Pilato, Kiosya, Lisi, Inshina & Biserov, 2011. This action leads to uniformization of the genus diagnosis from the morphological point of view, which brings it closer to being considered as monophyletic. Abstract Revisions and redescriptions of taxa described in the past and that are now categorized as insufficiently diagnosed often play a crucial role in making further progress in modern taxonomy in many groups of organisms. Here we revised an enigmatic tardigrade species Tenuibiotus hyperonyx (Maucci, 1983) based on the newly discovered topotypic population from the Italian Alps. We performed an integrative analysis of morphological and genetic data in order to present an upgraded species description and elucidate its phylogenetic position. Our results enabled us to confidently place T. hyperonyx within the family Richtersiusidae, as a member of the genus Diaforobiotus. This change, together with a re-assessment of microphotographs of the Tenuibiotus willardi (Pilato, 1977) and Tenuibiotus bozhkae Pilato, Kiosya, Lisi, Inshina & Biserov, 2011 types, led to the discussion on species composition with narrative taxa amendments for the taxonomic parties involved in the proposed alteration.

Macrobiotus hyperonyx Maucci, 1982 [40] was described from the highest mountain of the Sella Group (Piz Boè, 3152 m a.s.l) within the Dolomites (Italy), and so far, it is known only from its type locality. Due to the animal's morphology, it has been included within the informal tenuis-group by Maucci [41,42], which was later revised by Tumanov [43,44]. The morphological premises of the tenuis-type claw (a common tract distinctly longer compared to other macrobiotids and a short secondary branch forming an almost right angle with the primary branch) have been considered as a main diagnostic characteristic for the genus Tenuibiotus erected by Pilato and Lisi [45]. The genetic data for the genus Tenuibiotus are limited to the four taxa that were published in three scientific papers [29,46,47]. Only in the first work did the authors demonstrate the monophyly of these taxa in their extensive phylogenetic study on the family Macrobiotidae. However, it was noted that all these Tenuibiotus populations were morphologically uniform with a non-porous cuticle and two macroplacoids in the pharynx [29]. Therefore, as some of the taxa originally attributed to the genus exhibit pores in the cuticle or three macroplacoids in the pharynx, and these traits are known to have great importance for eutardigrade classification, Stec et al. [29] suggested Tenuibiotus with such taxa composition to be polyphyletic.
In this work, we rediscovered a topotypic population of Tenuibiotus hyperonyx (Maucci, 1982) [40] in the Dolomite Alps on which the integrative taxonomic analysis was performed. This included detailed morphological and morphometric examination under phase contrast and scanning electron microscopy (PCM and SEM, respectively) and sequencing of four molecular markers (18S rRNA, 28S rRNA, ITS-2 and COI). Given that T. hyperonyx exhibits pores in the body cuticle, we were able to test the hypothesis on Tenuibiotus polyphyly by elucidating its phylogenetic position. Additionally, the examination of microphotographs of the Tenuibiotus willardi (Pilato, 1977) [48] types enabled us to verify the presence of taxa possessing three macroplacoids in the pharynx within the genus.

Samples and Specimens
Four moss or moss + lichen samples containing T. hyperonyx were collected from rocks at the nival zone in Trento (the Dolomites, Piz Boè; all at ca. 3000 m a.s.l.) by Witold Morek and Katarzyna Vončina on 18 July 2020 ( Table 1). The samples were examined for tardigrades according to Stec et al. [49]. Other tardigrades present in the samples included Cornechiniscus holmeni (Petersen, 1951) [50], Echiniscus granulatus (Doyère, 1840) [51], and representatives of the genera Milnesium, Pseudechiniscus, and Richtersius. Extracted animals and eggs were divided into several groups used for different analyses, i.e., morphological analysis in PCM and SEM, as well as DNA sequencing (Table 1). Two additional specimens of Crenubiotus sp. from a Greenlandic moss sample (Sermersooq; 74 • 29 0.766 N, 20 • 32 18.308 W; 77 m a.s.l.; moss from soil; tundra; 07.2021; coll. Michał Kolasa) were sequenced in order to increase our phylogenetic dataset.  [52] with modifications described in detail in Stec et al. [35]. Each specimen was mounted in water and examined under light microscopy prior to DNA extraction. We sequenced four DNA fragments, three nuclear (18S rRNA, 28S rRNA, ITS2) and one mitochondrial (COI). All fragments were amplified and sequenced according to the protocols described in Stec et al. [35]; primers with original references are listed in Table 2. Sequencing products were read with the ABI 3130xl sequencer at the Molecular Ecology Lab, Institute of Environmental Sciences of the Jagiellonian University, Kraków, Poland. Newly obtained sequences were submitted to NCBI GenBank (see "Results" section and Table 3). Sequences in this study were processed and handle in BioEdit ver. 7.2.5 [53].
The sequences were aligned using the AUTO method (in the case of COI and ITS-2) and the Q-INS-I method (18S rRNA and 28S rRNA) in MAFFT version 7 [70,71] and manually checked against non-conservative alignments in BioEdit. Then, the aligned sequences were trimmed to 1009 (18S rRNA), 832 (28S rRNA), 543 (ITS-2) and 658 (COI) bp. All COI sequences were translated into protein sequences in MEGA7 version 7.0 [72] to check against pseudogenes. The sequences were then concatenated in SequenceMatrix [73]. Using PartitionFinder version 2.1.1 [74] under the Akaike Information Criterion (AIC), and with a greedy algorithm [75] implemented within the software, we chose the best scheme of partitioning and substitution models for posterior phylogenetic analysis. We ran the analysis to test all possible models implemented in MrBayes and RAxML software. As the COI is a protein-coding gene, before partitioning, we divided our alignment of this marker into three data blocks constituting three separated codon positions.
Bayesian inference (BI) marginal posterior probabilities were calculated using MrBayes v3.2 [76]. Random starting trees were used, and the analysis was run for 10 million generations, sampling the Markov chain every thousand generations. An average standard deviation of split frequencies of <0.01 was used as a guide to ensure the two independent analyses had converged. The program Tracer v1.6 [77] was then used to ensure Markov chains had reached stationarity and to determine the correct 'burn-in' for the analysis, which was the first 10% of generations. The ESS values were greater than 200 and a consensus tree was obtained after summarizing the resulting topologies and discarding the 'burn-in'. The maximum-likelihood (ML) tree was computed using RAxML v8.0.19 [78]. The strength of support for internal nodes of the ML construction was measured using 1000 rapid bootstrap replicates. All final consensus trees were visualized with FigTree v.1.4.3 available from (http://tree.bio.ed.ac.uk/software/figtree, accessed on 10 August 2018).

Microscopy and Imaging
Specimens for light microscopy were mounted on microscope slides following the protocol by Morek et al. [79]. Slides were examined under an Olympus BX53 light microscope with phase contrast (PCM), associated with an Olympus DP74 digital camera. Immediately after mounting the specimens in the medium, slides were checked under PCM for the presence of males and females in the studied population [61,80]. Specimens for the SEM analysis were processed according to the protocol by Stec et al. [49]. Bucco-pharyngeal apparatuses were extracted following the protocol of Eibye-Jacobsen [81] as modified by Gąsiorek et al. [82]. Specimens were examined under high vacuum in a Versa 3D DualBeam Scanning Electron Microscope (SEM) at the ATOMIN facility of the Jagiellonian University, Kraków, Poland. All figures were assembled in Corel Photo-Paint X6.

Morphometry and Morphological Nomenclature
All measurements are given in micrometers (µm). The sample size was adjusted following recommendations in Stec et al. [83]. Structures were measured only if their orientation was suitable. Body length was measured from the anterior extremity to the end of the body, excluding the hind legs. The terminology used to describe the oral cavity armature and egg-shell morphology follows Michalczyk and Kaczmarek [84] and Kaczmarek and Michalczyk [85]. The macroplacoid length sequence is given according to Kaczmarek et al. [86] whereas morphological states of the cuticular bars on legs follow Kiosya et al. [36]. The buccal tube length and the level of the stylet support insertion point were measured according to Pilato [87]. The pt index was calculated as the ratio of the length of a given structure to the length of the buccal tube expressed as a ratio [87]. Measurements of buccal tube widths, heights of claws heights and eggs follow Kaczmarek and Michalczyk [85]. The claw common tract index (cct) is the proportion of the height of the common tract of the claw (measured from the claw base to the separation point between the first and the second branch) to the total claw height expressed as a percentage [22].

Material Examined
In total, we examined 64 animals and 4 eggs. Specimens were mounted on microscope slides in Hoyer's medium (46 animals + 3 eggs), fixed on SEM stubs (14 + 1, including four bucco-pharyngeal apparatuses) and processed for DNA sequencing (4 animals); details on topotypic locality and specific samples are given in Table 1

Animals
When alive, body pale yellow to light orange (Supplementary Materials SM.04); after fixation in Hoyer's medium, body transparent ( Figure 2A). Large, black granular eyes present, also visible in specimens mounted in Hoyer's medium. Body cuticle smooth, without granulation but with circular or elliptical pores with uneven edges (0.8-2.0 µm in diameter) distributed randomly on the entire body cuticle with the largest pores present in the dorso-caudal cuticle ( Figures 2B,C and 3A,B). Pores on the ventral side of the body less frequent than on the dorsal side ( Figure 2B,C). Granulation on all legs absent ( Figures 4A-C and 5A-D). An obvious cuticular fold is present on the frontal side of each leg I-IV and clearly visible in PCM and SEM ( Figures 4A-C and 5A-D). The pulvini are present on each leg I-III on the internal leg surface and are almost indistinct in PCM but clearly visible in SEM ( Figure 5B). Claws slender, of the richtersiusid type. Common tract with a system of internal septa, and with an evident stalk connecting the claw to the lunula ( Figures 6A-C and 7A,B). The common tract nearly as long as the half of the entire claw height ( Figures 6A,C and 7A,B). Primary and secondary branches form almost a right angle when bifurcating ( Figures 6A,C and 7A,B). Primary branches with accessory points fitted tightly to the branch and thus indistinct in PCM but well visible only in SEM ( Figures 6A,C and 7A,B). Large, wide lunulae present on all legs and only in hind legs equipped with clearly visible teeth ( Figures 6A-C and 7A,B). A single continuous cuticular bar and paired muscle attachments present just above claws on legs I-III ( Figures 4B, 5A,B, 6B and 7C). In PCM, the cuticular bar is indented towards the lunulae, with shaded extensions towards muscle attachments, whereas in SEM, it is visible as a continuous (only sometimes constricted in the middle) thickening ( Figures 5A,B, 6B and 7C).
Mouth antero-ventral. Relatively short bucco-pharyngeal apparatus ( Figures 8A and 10A) with ten peribuccal lamellae, a rigid buccal tube, bended anteriorly, with the ventral lamina. Based on PCM observations, the oral cavity armature is poorly developed and composed only of the second and the third band of teeth ( Figure 8B-F). However, the first band is present and visible only in SEM and composed of very small granular teeth positioned just below peribuccal lamellae ( Figure 9A-C). In PCM, as well as in SEM, the second band of teeth is composed of several rows of granular teeth, of which the most posterior row comprises the larger teeth ( Figures 8B-E and 9B,C). The teeth of the third band are located within the posterior portion of the oral cavity, anteriorly to the buccal tube opening ( Figures 8B-F and 9A-C). The third band of teeth is divided into the dorsal and the ventral portion ( Figures 8B-F and 9A-C). The dorsal portion is composed of only one large tooth positioned in the very posterior portion of the oral cavity and far from the second band of teeth, whereas the ventral portion comprises small lateral ridges, between which a minute medial tooth is present ( Figures 8B-E and 9B,C). The ventral portion of the third band of teeth is especially faint in PCM ( Figure 8C,E). The porous areas are present in the buccal crown ( Figure 10B). In SEM, two depressions are visible on the ventral side of the buccal tube just below the stylet support insertion points ( Figure 10C). Typically shaped furcae with enlarged basal portion also exhibiting two depressed circular areas just above the two caudal branches (visible only in SEM; Figure 10D). Pharynx spherical, with triangular apophyses, three anterior cuticular spikes (typically only two are visible in any given plane) and two rod-shaped macroplacoids (2 < 1) (Figures 8G and 10E,F). The first macroplacoid is anteriorly narrowed and constricted in the middle, whereas the second has a sub-terminal constriction ( Figures 8G and 10E,F). Microplacoid absent. Measurements of animals and statistics are presented in Table 4.       Table 4. Measurements (in µm) of selected morphological structures of animals from the topotypic population of D. hyperonyx comb. nov. (Maucci, 1982) mounted in Hoyer's medium; N-number of specimens/structures measured, RANGE refers to the smallest and the largest structure among all measured specimens; SD-standard deviation. Pt values are given with italics.

Eggs
Laid freely, yellowish to light orange (Supplementary Materials SM.04), spherical with conical processes and smooth egg surface without areolation, reticulation or lightrefracting dots ( Figure 11A-D and Figure 12A-D). The process apices can sometimes exhibit faint nodular projection at the top ( Figure 12B-D). Distal portions of the processes are covered by faint granulation: Dark dots of rough/jagged wall in the process midsection (PCM)/clear hemispheres (SEM) ( Figure 11C,D and Figure 12A-D). The labyrinthine layer between the process walls as well as dark thickenings around process bases absent. Delicate micropores near the process bases rarely present and visible only in SEM ( Figure 12B-D). Egg measurements and statistics are presented in Table 5.   Table 5. Measurements (in µm) of selected morphological structures of the eggs from the topotypic population of D. hyperonyx comb. nov. (Maucci, 1982) mounted in Hoyer's medium; all three eggs were damaged in permanent slides, thus the diameter and number of processes on the egg circumference cannot be measured/counted; N-number of eggs/structures measured, RANGE refers to the smallest and the largest structure among all measured specimens; SD-standard deviation.

Reproduction
The examination of adults freshly mounted in Hoyer's medium revealed testes filled with spermatozoa in each of the four examined samples, confirming the species to be dioecious ( Figure 13A,B). Any other secondary sexual phenotypic characters, e.g., gibbosities on the hind legs in males, absent.

Discussion
Morphological information gathered in our integrative study on the newly discovered population, compared also with type specimens, fully support its identification as Tenuibiotus hyperonyx (Maucci, 1982) [40]. Based on the recovered phylogenetic position within the family Richtersiusidae as well as its phenotypic affinity to the genus Diaforobiotus, the species is proposed to be transferred to the later taxon. The proposed change requires amendments to the diagnosis of the family Richtersiusidae, which, among other characters, is now also defined by the presence of large teeth on all lunulae. Since Diaforobiotus hyperonyx comb. nov. exhibits teeth only in lunulae in the hind legs, the for-mer character cannot be exclusive for the family. This further brings our attention to the two recent papers focusing on the phylogenetic relationships between four genera, namely Diaforobiotus, Richtersius Pilato & Binda, 1989 [94], Adorybiotus Maucci & Ramazzotti, 1981 [95] and Crenubiotus. Both these papers [11,27] were published at approximately the same time and came up with different interpretations of the relationship between the mentioned taxa. Guidetti et al. [11] studied the phylogenetic position of the newly discovered Crenubiotus species, also pinpointing its classification within the family Richtersiusidae, whereas Stec et al. [27] phylogenetically analyzed two different Crenubiotus and one additional Adorybiotus populations. Both studies recovered the sister relationships between clades Adorybiotus + Crenubiotus and Murrayon + Dactylobiotus, with Richtersius + Diaforobiotus being a sister clade to this entire cluster [11,27]. However, only Stec et al. [27] proposed splitting the family Richtersiusidae and erected the new family Adorybiotidae that comprises Adorybiotus and Crenubiotus, with the main distinctive character being the absence and presence of microplacoid in these families, respectively [27]. The phylogenetic analysis with increased taxa sampling conducted in our study yielded even higher support for the scenario proposed by Stec et al. [27], further confirming the validity of the family Adorybiotidae ( Figure 1, Supplementary Materials SM.02). Finally, it would be worth discussing the obvious elongation of the claw primary branches in the Diaforobiotus hyperonyx comb. nov., which make it distinct from all other Diaforobiotus populations recorded so far. This character could potentially constitute a clear diagnostic trait for a putative new genus, especially together with the recovered sister relationship between this species and remaining Diaforobiotus taxa (Figure 1). Nevertheless, we believe that, currently, the genus erection would be premature as the phylogenetic sampling of Diaforobiotus taxa is still scarce and the genus likely comprises at least several other species. Furthermore, the claw elongation in macrobiotids was recently reported to be caused most probably by the wet and icy environment [96]. The authors presented the phylogeny of the family Macrobiotidae demonstrating convergent evolution in claw elongation in the Macrobiotus ariekammensis complex and Mesobiotus barabanovi (Tumanov, 2005) [43], with both of them being deeply nested within their respective genera. The occurrence of Diaforobiotus hyperonyx comb. nov. in the high mountains (Dolomite Alps) further supports the hypothesis of environmental factors affecting the claw phenotypic changes in macrobiotids, constituting the third example of convergently evolving claws phenotypes within Macrobiotoidea.
As mentioned in the Introduction, although the genus Tenuibiotus was recovered to be monophyletic in the phylogeny presented by Stec et al. [29], it was suggested to still be polyphyletic due to the morphological heterogeneity of the included taxa at that time. The mixed morphological characters that led to such a suggestion were: (i) The presence or absence of cuticular pores and (ii) varying numbers of placoids in the pharynx. After transferring T. hyperonyx to the genus Diaforobiotus, all remaining taxa currently recognized in the genus Tenuibiotus (13 species) exhibit a non-porous cuticle, which signifies that this trait is a solid and uniform diagnostic character of the genus. The nomenclatural and classification change proposed by us also provided the second uniform morphological trait that characterizes all members of the genus Tenuibiotus-the presence of a microplacoid in the pharynx. Regarding the number of macroplacoids, the majority of genus members exhibit two macroplacoids in the pharynx, excluding two species, which were reported to have three macroplacoids. These are T. willardi and T. bozhkae. The original description of the first one reports three or two macroplacoids to be present in the type population [48]. However, the re-examination of the holotype and paratype bucco-pharyngeal apparatuses confirmed the presence of two macroplacoids in the pharynx ( Figure 14A-C). Since the name-bearing specimen exhibits this trait and it is considered important in the tardigrade classification, it should be considered the primary character state of the nominal species. The original description of the second species indeed reports three macroplacoids [89] and, at the same time, indicates morphological similarity with Tenuibiotus ciprianoi (Guil, Guidetti & Machordom, 2007) [97]. Interestingly, the latter species exhibits two macroplacoids in the pharynx, with the first one being extremely deeply constricted in the middle ( [97]; Figure 3B). Although the quality of morphological documentation is much better in Guil et al. [97], the comparison of microphotographs presented in both these papers reveals that there is no obvious difference between them in this particular character ( [89,97], especially the comparison of Figures 2B and 3B, respectively). The examination of additional microphotographs of the holotype and paratype of T. bozhkae ( Figure 14E-G) also did not allow us to indicate a clear difference between this species and T. ciprianoi. Thus, we consider T. bozhkae to possess only two macroplacoids in the pharynx, with the first one being deeply constricted. Therefore, we propose a third, uniform diagnostic morphological character for the genus Tenuibiotus, which is the presence of two macroplacoids in the pharynx.

Conclusions
Our work is yet another example of the great value of the integrated approach at a taxonomic and phylogenetic level when studying groups of small organisms such as meiofauna that are known to have a limited number of informative morphological characteristics. In this case, the revision of one enigmatic species and the discussion induced thereafter led to important amendments for other tardigrade taxa. We greatly endorse continuous studies on tardigrade taxonomy that implement the analyses of detailed phenotypic information tightly linked to genetic data. Surely, these will not only bring discoveries of new, exciting taxa but will also help to elucidate the trajectories of morphological evolution within this group of microscopic animals.

Data Availability Statement:
The author confirms that the data supporting the findings of this study are available within the article and its Supplementary Materials. The DNA sequences generated in this study are available in GenBank.